Assessing the potential molecular mechanism of arsenite-induced skin cell senescence

Abstract

Arsenic exposure is a public health concern worldwide. Skin damage, as a typical lesion of arsenic exposure, the mechanism is still unknown. Studies have found that cellular senescence plays a key role in arsenic-induced skin damage, and the previous research found that the ERK/CEBPB signaling pathway may be an important molecular event of arsenic-induced skin cell senescence, but its specific mechanism is unknown. In this study, genetic engineering technology was used to construct stable HaCaT cell lines, and the role and mechanism of ERK/CEBPB signaling pathway in arsenic-induced HaCaT cell senescence were verified by knockdown and overexpression of ERK and CEBPB in both forward and backward. It was found that knockdown of CEBPB or ERK can downregulate the ERK/CEBPB signaling pathway and reduce arsenic-induced skin cell senescence. In contrast to knockdown, overexpression of CEBPB or ERK can upregulate the ERK/CEBPB signaling pathway and aggravate the senescence of skin cells caused by arsenic. These findings suggest that sodium arsenite can further promote SASP secretion and the expression of p53, p21 and p16 ^INK4a by activating the ERK/CEBPB signaling pathway, induce cell cycle arrest and trigger cellular senescence.

Introduction

Low arsenic exposure is a public health concern worldwide, with more than 2.75% of the global population exposed to arsenic.¹ Skin damage is a typical lesion in patients with arsenic poisoning,² and long-term exposure to arsenic can lead to excessive keratosis of the palms and plantars, skin pigmentation, skin diseases such as Bowen’s disease, and even malignant tumors.^3–5 However, the pathogenic mechanism of arsenic-induced skin injury needs to be further studied.

There are two main features of cellular senescence, namely cell cycle arrest and the Senescence-associated secretory phenotype (SASP).^6,7 Senescence is not only a major driver of many chronic diseases,^6,8–11 but also helps the body fight tumorigenesis.^12,13 In view of these antagonistic effects, cellular senescence is seen as a double-edged sword for health and disease.¹⁴ Senescence-associated β-galactosidase (SA-β-gal) is by far the most specific senescence-related marker that can identify cellular senescence, and once cells are senescated, they are accompanied by an increase in SA-β-gal levels.^15,16 The key role of cellular senescence in arsenic poisoning has been noted. Studies have found that arsenic can increase SA-β-gal activity, upregulate the expression of p53, p21, and p16^INK4a, and promote the secretion of SASP.^17–19 More importantly, the relationship between cellular senescence and arsenic-induced skin damage was further validated through p16^INK4a reporter mouse experiments and case-control studies from India.^17,19 However, the mechanism of arsenic-induced skin aging still needs further study. In the process of arsenic-induced skin damage, it is accompanied by oxidative stress, immune inflammation and DNA damage response,^20,21 which are important factors that cause cell aging.^22–25 In addition, the previous research of our group found that the ERK/CEBPB signaling pathway may be an important molecular event of arsenic-induced skin cell aging,²⁶ but its specific mechanism needs to be further confirmed.

In summary, we constructed HaCaT cell lines with knockdown and overexpression of ERK and CEBPB based on previous research by our predecessor and research group. We observed ERK1, ERK2, and CEBPB signaling pathway-related indexes, as well as cell senescence-related indexes [SA-β-gal, cell cycle, SASP (IL-1α, IL-6, IL-8, TGF-β1, MMP-1, MMP-3, VEGF) secretion levels and the mRNA and protein expression levels of cell cycle-related indexes p53, p21, and p16^INK4a] in the control group, sodium arsenite (NaAsO₂) group, no-load group (NC), NaAsO₂ + transfection group. Our aim is to investigate the role and mechanism of the ERK/CEBPB signaling pathway in arsenic-induced HaCaT cell senescence, and better explain the possible mechanism of sodium arsenite-induced skin cell senescence. The results of this study provide experimental evidence and contribute to the understanding of the arsenic-induced skin damage mechanism.

Materials and methods

Cell culture and treatments

HaCaT cells were kindly provided by a Cell Bank, the Kunming Academy of Sciences in China, and cultured in DMEM (Gibco, C11995500BT) containing 10% FBS (Gibco, 10099141) and 1% puromycin (Solarbio, P8230) at 37 °C with 5% CO₂. In this study, HaCaT cells were used to construct an arsenic exposure model, and other HaCaT cell lines of knockdown and overexpression of CEBPB and ERK (Guangzhou Huiqi Biotechnology Co., Ltd) were constructed by genetic engineering technology, and the culture methods and identification results of stable cells were shown in Supplementary document 1 (Online Resource 1) and Supplementary Fig. 1a (Online Resource 1). The source and culture methods of the cell lines were carried out as described above,²⁶ recombinant lentivirus plasmids were constructed for knocking down or over-expressing the target gene by cloning different shRNA and target gene CDS fragments into various plasmid vectors. Lentivirus was then used to infect HaCaT cells, and stable knockdown and overexpression cell lines were obtained by drug screening. NaAsO₂ was purchased from Merck in Germany.

The design of this study continues the previous experiments of the research group.²⁶ A population-based 20-year follow-up study in a unique coal-borne arsenicosis County in Guizhou, China.²⁷ The results showed that arsenic concentration in local external environmental medium and total arsenic uptake by local residents were both much higher in the past, although this uptake has dropped to a very low level currently. Additionally, the World Health Organization recommends that arsenic concentrations in drinking water be below 10 micrograms per liter (0.13 M). Considering the reference value recommended by the World Health Organization, in order to better simulate the actual situation of current low arsenic exposure in arsenic poisoning areas, we chose low concentrations of arsenic to treat HaCaT cells in vitro. We selected NaAsO₂ concentrations of 0.05 μM, 0.1 μM, and 0.25 μM for HaCaT cells to detect SA-β-gal activity of HaCaT cells after arsenic exposure. The stable cell culture method described above treated cells with 0.00, 0.25 μM NaAsO₂ for 72 h,²⁶ including: control group, NaAsO₂ group, NC group, transfection+NaAsO₂ group.

Quantitative real-time PCR

As previously described in the literature,²⁶ after RNA extraction by Trizol (purchased from Invitrogen), then cDNA was obtained using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, RR047A) according to the manufacturer’s instructions, and finally the BioRad CFX96 machine (BIO-RAD, USA) was used to detect the relative expression levels of mRNA of ERK1, ERK2, CEBPB, p53, p21, p16^INK4a according to cyclic thresholds, β-actin as the internal reference gene. Supplementary Fig. 1b (Online Resource 1) shows that the primers used in this experiment were synthesized by Generay Bioengineering Co., Ltd (China).

Western blotting

As described in previous studies,²⁶ cells were collected and the prepared protein lysate [RIPA:PMSF:phosphatase inhibitor cocktail = 100:1:2, all from Solaibo (China)] was used for total protein extraction, followed by protein quantification, and finally protein electrophoresis, membrane transfer, and incubation of the corresponding primary antibodies [ERK1 (1:1,000, ab32537), ERK2 (1:1,000, ab32081), P-ERK (1:1,000, ab201015), CEBPB (1:1,000, ab32358), p21 (1:1,000, ab109520), p16^INK4a (1:1000, ab108349), and p53 (1:500, ab154036), these antibodies were purchased from Abcam (USA)] and secondary antibodies [Goat Anti–Rabbit IgG (H + L) HRP (#s0001), Goat Anti–Mouse IgG (H + L) HRP (#s0002), these antibodies were purchased from Affinity (China)]. The secondary antibody concentration P-ERK is 1:100,000, CEBPB is 1:20,000, p21 and p16^INK4a are 1:1,000, and others are 1:10,000. measured using the chemiluminescence imaging system of Bio-Rad (ChemiDoc, USA). ImageJ software was used for gray value analysis, and the ratio of the gray value of the target protein and the reference protein GAPDH [1:10,000, Bioworld (China)] was used to reflect the relative expression of the protein and normalize the blotting.

Flow cytometry assay

The same as described earlier,²⁶ the cell cycle was measured using the propidium iodide cell cycle assay kit from KeyGEN Biotech in China, as directed: cells were collected, fixed, then stained, and finally different cell cycles were determined using flow cytometry (FACSCelesta, BD, USA), and the percentages of cells G1, G2, and S phases were analyzed using FlowJo 10.3 software.

Enzyme-linked immunosorbent assays

Detect human-specific IL-1α, IL-6, IL-8, TGF-β1, MMP-1, MMP-3, and VEGF in cell supernatants using the R&D Quantikine(R) kit (Bio-Techne China Co. Ltd). The methodology for the above indicators is the same as in previous studies.²⁶ The study was designed with parallel samples and each sample was repeated 3 times. Finally, the average absorbance is calculated to assess the concentration of SASP.

Senescence-associated β-galactosidase assays

SA-β-gal activity of arsenic-exposed cells was detected using the Senescence β-Galactosidase Staining Kit (Beyotime, China). Prepare the staining solution according to the reagent instructions, incubate overnight at 37 °C in a CO₂-free incubator. The senescent cells stained in blue were counted under light microscopy (200×) and calculated the proportion of senescent cells in the total cells.

Statistical analysis

Adoption of SPSS 25.0 software for statistical analysis, data after normal distribution test, use one-way analysis of variance (ANOVA) to compare the differences between the groups. Further comparison between groups, LSD method was used if the variance was aligned; If the variance is uneven, Dunnett’s T3 method is used. The test level α = 0.05. A total of three repeated experiments and three replicates in each group were used in this study.

Results

NaAsO₂ can induce skin cell senescence and enhance SA-β-gal activity of skin cells

In the early stage, our research team found that arsenic can up-regulate the expression of cyclin, induce cell cycle arrest in G1 phase and promote the secretion of SASP.²⁶ To further verify the relationship between arsenic exposure and cell senescence, the activity of SA-β-gal was detected in this arsenic exposure experiment (Fig. 1a). It was found that SA-β-gal activity increased dependently with the dose and duration of arsenic exposure. Combined with previous findings, these results provide further evidence for arsenic-induced skin cell senescence. We can see that with the increase of infection concentration and infection time, the number of cells dyed blue gradually increases (Fig. 1c). It can be seen from (Fig. 1b) that compared with the control group, the activity of SA-β-gal in 0.10 and 0.25 μM NaAsO₂ treatment groups increased significantly (P < 0. 05), except in the 0.05 μM NaAsO₂-exposed group (P > 0.05); and the activity of SA-β-gal in 0.25 μM NaAsO₂ treatment group increased most obviously for 72 h.

Fig. 1.

Sodium arsenite can enhance SA-β-gal-positive of skin cells. All data are presented as mean ± standard deviation, n = 3 per group. ^*P < 0.05. a) Different doses and different times of arsenic exposure treated cells. b) Statistical results of SA-β-gal-positive cells at different arsenic exposure times. c) SA-β-gal positive cell detection at different arsenic exposure times.

Knockdown of CEBPB can reduce the secretion of SASP and reduce NaAsO₂-induced skin cell senescence

To verify the role of the transcription factor CEBPB in arsenic-induced HaCaT cell senescence, knockdown of CEBPB cell lines were constructed. It was found that knockdown of CEBPB could reduce the expression of genes related to cell cycle regulation and the secretion of SASP by downregulating ERK/CEBPB signaling pathway, alleviate cell cycle arrest, and then reduce cell senescence. The relative expression of mRNA and protein of CEBPB (Fig. 2g-–i), p53, p21 and p16^INK4a (Fig. 2j–l) in the NaAsO₂ + CEBPB-shRNA group, as well as the secretion of IL-1α, IL-6, IL-8, MMP-1, MMP-3, VEGF (Fig. 2b) and SA-β-gal (Fig. 2c and d) activities were significantly lower than those in the NaAsO₂ group (P < 0.05), the expression of ERK1, ERK2 and P-ERK (Fig. 2g–i) was not significantly different from that in the NaAsO₂ group. The NaAsO₂ + CEBPB-shRNA group exhibited a notable decrease in the proportion of G1 phase cells (Fig. 2e and f) and a corresponding increase in the proportion of S phase cells (Fig. 2e and f) compared to the NaAsO₂ group (P < 0.05).

Fig. 2.

Knockdown of CEBPB can reduce the key molecules in ERK/CEBPB signaling pathway, the expression of cycle-related proteins, cell cycle arrest, SASP secretion, and SA-β-gal activity. All data are presented as mean ± standard deviation, n = 3 per group. ^*P < 0.05. a) Knockdown/overexpression of CEBPB can promote/inhibit the expression of CEBPB, reduce/increase the expression of genes related to cell cycle regulation and the secretion of SASP, alleviate/aggravate cell cycle arrest, and then alleviate/aggravate cell aging. b) SASP after knockdown of CEBPB. c) Statistical results of SA-β-gal-positive cells after knockdown of CEBPB. d) SA-β-gal-positive cells after knockdown of CEBPB. e) Statistical results of the cell cycle after knockdown of CEBPB. f) The cell cycle after knockdown of CEBPB. g) Expression of genes associated with ERK/CEBPB signaling pathway after knockdown of CEBPB. h) Western blotting of ERK/CEBPB signaling pathway-related proteins after knockdown of CEBPB. i) Expression of ERK/CEBPB signaling pathway-related proteins after knockdown of CEBPB. j) Relative expression of p53, p21, and p16^INK4a mRNA after knockdown of CEBPB. k) Western blotting of p53, p21, and p16^INK4a after knockdown of CEBPB. l) Relative expression of p21 and p16^INK4a proteins after knockdown of CEBPB.

Overexpression of CEBPB can promote the secretion of SASP and aggravate NaAsO₂-induced skin cell senescence

To further explore the role of CEBPB in arsenic-induced HaCaT cell senescence, we constructed an overexpression of CEBPB cell lines. The results demonstrated that CEBPB overexpression was associated with upregulation of genes involved in cell cycle regulation and increased SASP secretion. Furthermore, CEBPB overexpression exacerbated G1 phase blockade and promoted cellular senescence. The relative expression of mRNA and protein of CEBPB (Fig. 3f–h), p53, p21, and p16^INK4a (Fig. 3i–k) in the NaAsO₂ + CEBPB-over group, as well as the secretion of IL-1α, IL-6, IL-8, TGF-β1, MMP-1, MMP-3, VEGF (Fig. 3a) and SA-β-gal (Fig. 3b and d) activities were significantly higher than those in the NaAsO₂ group (P < 0.05). Contrary to the results of CEBPB knockdown, the percentage of G1 phase cells significantly increased in the NaAsO₂ + CEBPB-over group (Fig. 3c and e), while the percentage of S phase cells decreased significantly compared to the NaAsO₂ group (P < 0.05). The expression of ERK1, ERK2, and P-ERK (Fig. 3f–h) in the NaAsO₂ + CEBPB-over group was also not significantly different from that in the NaAsO₂ group.

Fig. 3.

Overexpression of CEBPB can promote the key molecules in ERK/CEBPB signaling pathway, the expression of cycle-related proteins, cell cycle arrest, SASP secretion, and SA-β-gal activity. All data are presented as mean ± standard deviation, n = 3 per group. ^*P < 0.05. a) SASP after overexpression of CEBPB. b) Statistical results of SA-β-gal-positive cells after overexpression of CEBPB. c) Statistical results of the cell cycle after overexpression of CEBPB. d) SA-β-gal-positive cells after overexpression of CEBPB. e) The cell cycle after overexpression of CEBPB. f) Expression of ERK/CEBPB signaling pathway-related genes after overexpression of CEBPB. g) Western blotting of ERK/CEBPB signaling pathway-related proteins following overexpression of CEBPB. h) Expression of ERK/CEBPB signaling pathway-related proteins after overexpression of CEBPB. i) Relative expression of p53, p21, and p16^INK4a mRNA after overexpression of CEBPB. j) Western blotting of proteins p53, p21, and p16^INK4a after overexpression of CEBPB. k) Relative expression of p21 and p16^INK4a proteins after overexpression of CEBPB.

Knockdown of ERK can reduce the expression of CEBPB, which in turn reduces NaAsO₂-induced skin cell senescence

To verify the role of ERK/CEBPB signaling pathway activation in arsenic-induced HaCaT cell senescence, ERK knockdown cell lines were constructed. The results showed that ERK knockdown could downregulate the ERK/CEBPB signaling pathway, reduce the expression of genes related to cell cycle regulation and the secretion of SASP, alleviate cell cycle arrest, and reduce cell senescence. The relative expression of mRNA and protein of ERK1, ERK2, CEBPB (Fig. 4g–i), p53, p21, and p16^INK4a (Fig. 4j–l) in the ERK-shRNA+NaAsO₂ group, as well as the secretion of IL-1α, IL-6, IL-8, TGF-β1, MMP-1, MMP-3, VEGF (Fig. 4b) and SA-β-gal (Fig. 4c and d) activities were significantly lower than those in the NaAsO₂ group (P < 0.05). Compared with the NaAsO₂ group, the G1 phase (Fig. 4e and f) cells in the NaAsO₂ + CEBPB-shRNA group were significantly reduced, but the S (Fig. 4e and f) phase cells were significantly increased (P < 0.05).

Fig. 4.

Knockdown of ERK can downregulate ERK/CEBPB signaling pathway the, expression of cycle-related proteins, cell cycle arrest, SASP secretion, and SA-β-gal activity. All data are presented as mean ± standard deviation, n = 3 per group. ^*P < 0.05. a) Knockdown/overexpression of ERK can downregulate/upregulate ERK/CEBPB signaling pathway, inhibit/promote the expression of genes related to cell cycle regulation and the secretion of SASP, alleviate/aggravate cell cycle arrest, and then alleviate/aggravate cell aging. b) SASP after knockdown of CEBPB. c) Statistical results of SA-β-gal-positive cells after knockdown of ERK. d) SA-β-gal-positive cells after knockdown of ERK. e) Statistical results of the cell cycle after knockdown of ERK. f) The cell cycle after knockdown of ERK. g) Expression of genes associated with ERK/CEBPB signaling pathway after knockdown of ERK. h) Western blotting of ERK/CEBPB signaling pathway-related proteins after knockdown of ERK. i) Expression of ERK/CEBPB signaling pathway-related proteins after knockdown of ERK. j) Relative expression of p53, p21, and p16^INK4a mRNA after knockdown of ERK. k) Western blotting of p53, p21, and p16^INK4a after knockdown of ERK. l) Relative expression of p21 and p16^INK4a proteins after knockdown of ERK.

Overexpression of ERK can upregulate the expression of CEBPB, thereby aggravating NaAsO₂-induced skin cell senescence

In order to verify the role of ERK/CEBPB signaling pathway activation in arsenic-induced HaCaT cell senescence from both positive and negative directions, we constructed an overexpressed ERK cell line. The results showed that contrary to the results of knockdown of ERK, overexpression of ERK could upregulate ERK/CEBPB signaling pathway, promote the expression of genes related to cell cycle regulation and the secretion of SASP, aggravate the blockade of cell cycle in G1 phase, and aggravate cell senescence. The relative expression of mRNA and protein of ERK1, ERK2, CEBPB (Fig. 5f–h), p53, p21, and p16^INK4a (Fig. 5i–k) in the NaAsO₂ + ERK-over group, as well as the secretion of IL-1α, IL-6, IL-8, TGF-β1, MMP-1, MMP-3, VEGF (Fig. 5a) and SA-β-gal (Fig. 5a and d) activities were significantly higher than those in the NaAsO₂ group (P < 0.05). Contrary to the knockdown ERK results, compared with the NaAsO₂ group, the G1 phase (Fig. 5c and e) cells in the NaAsO₂ + ERK-over group were significantly increased, but the S phase (Fig. 5c and e) cells were significantly decreased (P < 0.05).

Fig. 5.

Overexpression of ERK can upregulate ERK/CEBPB signaling pathway, the expression of cycle-related proteins, cell cycle arrest, SASP secretion, and SA-β-gal activity. All data are presented as mean ± standard deviation, n = 3 per group. ^*P < 0.05. a) SASP after overexpression of ERK. b) Statistical results of SA-β-gal-positive cells after overexpression of ERK. c) Statistical results of the cell cycle after overexpression of ERK. d) SA-β-gal-positive cells after overexpression of ERK. e) The cell cycle after overexpression of ERK. f) Expression of ERK/CEBPB signaling pathway-related genes after overexpression of ERK. g) Western blotting of ERK/CEBPB signaling pathway-related proteins following overexpression of ERK. h) Expression of ERK/CEBPB signaling pathway-related proteins after overexpression of ERK. i) Relative expression of p53, p21, and p16^INK4a mRNA after overexpression of ERK. j) Western blotting of proteins p53, p21, and p16^INK4a after overexpression of ERK. k) Relative expression of p21 and p16^INK4a proteins after overexpression of ERK.

Discussion

Arsenic is a widespread environmental toxicant, and long-term exposure to inorganic arsenic is a major environmental public health problem worldwide.²⁸ As one of the main organs of arsenic toxicity, the skin is also the earliest site of lesions.^4,29 As we all know, arsenic mediates skin damage through oxidative stress,^20,21 immune inflammation and DNA damage,^20,21 which are thought to be important factors in cellular senescence.^22–25

A population-based study in Chile suggests that arsenic exposure in the early stages of life may be associated with biological aging in adulthood.³⁰ Other studies have shown that arsenic can cause cellular senescence.^17–19,31 Cellular senescence occurs with cell cycle arrest and increased SASP secretion, accompanied by the expression of aging-related genes and proteins.^6,7 Previous studies have found that arsenic can promote the expression of p53, p21, p16^INK4a, and SASP in skin cells,²⁶ and observed cell cycle arrest in the G1 phase, which is consistent with the above findings. The results in this section provide limited evidence that arsenic can induce HaCaT cell senescence. During the senescence of cells, they are accompanied by an increase in SA-β-gal levels.^15,16 In order to further verify that arsenic can induce skin cell senescence, this study further detected on the basis of previous studies and found that arsenic can enhance the SA-β-gal activity of skin cells, and there is a significant dose and time-effect relationship between arsenic and SA-β-gal activity. This result provides further evidence that arsenic can induce skin cell senescence.

Stimulated by extracellular stress conditions, MAPK is activated to control cell cycle arrest through p53/p21, p16/pRB signaling pathways.⁶ As ERK is one of the main members of MAPK, and arsenic can activate ERK,^32–34 we have reason to suspect that the ERK signaling pathway is involved in the secretion of SASP and plays an important role in cell cycle arrest. Our study revealed that knockdown of ERK led to the downregulation of the ERK/CEBPB signaling pathway. This was accompanied by a reduction in arsenic-induced secretion of SASP, as well as decreased expression of cycle-related proteins such as p53, p21 and p16^INK4a. Additionally, cell cycle arrest was alleviated, resulting in a decrease in the number of G1 phase cells caused by NaAsO₂ and an increase in the number of S phase cells. However, the overexpression of ERK represents the opposite of ERK knockdown. The results of this study provide evidence for the above assumptions.

Previous results suggest that ERK can regulate the downstream transcription factor CEBPB through phosphorylation,^35–37 and previous results provide some evidence for this hypothesis.²⁶ Our study revealed that knockdown and overexpression of CEBPB did not significantly affect ERK expression levels. However, knockdown and overexpression of ERK led to corresponding changes in the expression levels of both ERK and P-ERK. Specifically, ERK knockdown resulted in decreased mRNA and protein expression of CEBPB, whereas ERK overexpression led to increased mRNA and protein expression of CEBPB. These findings further support the conclusion that ERK regulates the downstream transcription factor CEBPB through phosphorylation. Moreover, this study found that after knockdown ERK or CEBPB, arsenic-induced SA-β-gal activity of HaCaT cells was decreased; after overexpression of ERK or CEBPB, arsenic-induced SA-β-gal activity of HaCaT cells was enhanced. This finding provides further supporting evidence for the ERK/CEBPB signaling pathway to participate in low-dose arsenic-induced skin cell senescence. Although our study provides insight into the role of the ERK/CEBPB signaling pathway in vitro, further research is required to determine whether this pathway plays a similar role in animal models and human health. Additionally, the targeted regulation of ERK on CEBPB can be analyzed through co-immunoprecipitation assays.

CEBPB was identified as a regulator of SASP,³⁸ which regulates the secretion of SASP involved in cellular senescence. There are also studies that show that SASP is regulated by the MAPK signaling pathway.³⁹ After knockdown of ERK or CEBPB, we observed a decrease in the secretion of arsenic-induced SASP, while in overexpression of ERK or CEBPB, the secretion of arsenic-induced SASP increased. The above results further verify that “CEBPB can regulate SASP secretion”. For the conclusion that “SASP is regulated by MAPK signaling pathway”, combined with “ERK can regulate the downstream transcription factor CEBPB by phosphorylation”, we suspect that it may be because the activation of ERK in MAPK further activates CEBPB and leads to increased SASP secretion. These findings will contribute to a deeper understanding of the mechanisms of arsenite-induced skin cell senescence.

In summary, our results provide limited evidence for the conclusion that sodium arsenite can further promote SASP secretion and expression of p53, p21, and p16^INK4a by activating ERK/CEBPB signaling pathway, induce cell cycle arrest, and trigger cell senescence. In addition, relevant departments should continue to adhere to the post-management of prevention and control in the ward to ensure the health of people in arsenic poisoning areas.

Author contributions

All authors contributed to the study conception and design. Sufei Du Performed the Experiments, Data Curation and Writing—original draft preparation. Fan Yang, Liping Wu, Dexiu Hu, Yuhong Zhang, Maoyuan Gong, Yang Yang and Xingcan Yang performed the experiments. Qibing Zeng was responsible for the design and supervision of this research, and participated in the drafting of the article, critically revising the important knowledge content. All authors have read and agreed to the published version of the article.

Funding

This work was supported by the National Natural Science Foundations of China (No. 82360631, 81860569), Guizhou Province Science and Technology Plan Project of China (No. ZK [2021] key 009), China Postdoctoral Science Foundation (No. 2020M683375), and the Excellent Young Talents Plan and High–Level Talents Startup Fund Project of Guizhou Medical University (No. [2021]102 and J [2020]075).

Conflict of interest statement: None declared.

Data availability

The datasets used and analyzed during this study are available from the corresponding author on reasonable request.